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. 2018 Feb 17;26(7):1644–1659. doi: 10.1016/j.ymthe.2018.02.012

Figure 3.

Figure 3

In Vivo Transplantation of HVPs under Renal Capsule

(A) Schematic illustration of HVP generation, differentiation, and transplantation. HVPs were injected under the kidney capsule or intra-myocardially into the heart (injection site marked with squares); photos illustrate where the HVP graft was formed 2 months after transplantation, when it was recovered. (B) Electron microscopy of the HVP patch in the kidney capsule revealed mature ultra-structure, including organized and aligned sarcomeres, with well-defined Z-line, M-line, A-band, and I-band (left micrograph). Also apparent were desmosomes, mitochondria, myofilament bundles (middle panel), and capillaries containing red blood cells (right panel). (C) High-resolution immunofluorescence images further demonstrate sarcomeric organization within HVP grafts, with transverse orientation of a-actinin at z-lines and actin staining between z-lines. Membrane staining (WGA) revealed the presence of developing T-tubules (middle panels, high magnification of indicated region at right). (D) Immunofluorescence staining of the HVP kidney patch, with antibodies against MLC2a, MLC2v, cTnT, aSMA, ISL1, laminin, HCN4, human fibronectin, VE-cadherin, and Ki67. Scale bar, 200 μm. Red lectin was injected via the tail vein, illustrating a direct connection of the HVP kidney patch with the host venous system. Scale bar, 50 μm. (E and F) A 6+-week-old HVP kidney patch was harvested for ex vivo optical mapping and observed under brightfield (E) to be electrically responsive to stimulation (F). (G) A unique developmental window for HVP engraftment occurred at day 6 of differentiation; day 3 mesodermal MESP1+ cells resulted in teratomas, and day 10 late NKX2.5+ heart precursors failed to form a ventricular muscle graft patch (n = 4). (H and I) Cross-sectional area of a 6+-week-old HVP kidney graft patch during relaxation (i.e., diastolic phase; H) and maximal contraction (i.e., systolic phase; I), respectively, as imaged by ultrasound. (J) Measured cross-sectional area of the graft patch during diastole and systole.